| Background and Objective: Diabetes mellitus(DM)is a common endocrine and metabolic disease,while diabetic neuropathic pain(DNP)is one of the most common chronic complications of diabetes and one of the most common types of clinical neuropathic pain.The pathogenesis of DNP is currently unclear,and the treatment is not ideal,seriously affecting the quality of life of patients.Studies have shown that dorsal root ganglion(DRG)satellite glial cells(SGCs)P2X7 receptors are involved in the pathogenesis of neuropathic pain and inflammatory pain.Neurons transient receptor potential vanilloid 1(TRPV1),also known as capsaicin receptor,is closely related to diabetic neuropathic pain.And studies have shown that there is an interaction between neurons and glial cells in sensory ganglia.Based on the above,it is suggested that DRG satellite glial cell P2X7 receptor and neuron TRPV1 may have a certain relationship and role in diabetic neuropathic pain.Therefore,this study aims to explore the role of DRG satellite glial cell P2X7 receptor in neuropathic pain in a rat diabetic model mediated by neuron TRPV1 and its possible mechanism,which will provide a new way for research on the pathogenesis and related prevention and treatment of diabetes complicated with neuropathic pain.Methods: In this experiment,DNP model rats were established by high-sugar and high-fat diet plus low-dose STZ injection.Experimental groups: control group(Control),DNP rat model group(DNP),DNP rat model plus A438079 treatment group(DNP+A438079)and DNP rat model plus normal saline control group(DNP+NS,NS means normal saline),6 rats per group.Experimental content:determination of blood glucose and body weight of experimental rats;observe the changes of the mechanical hyperalgesia and thermal hyperalgesia in each group of experimental rats through behavioral testing;use real-time fluorescent quantitative PCR(q PCR)technology to detect changes in the levels of TRPV1 and P2X7 m RNA in rat DRG;detect the expression of TRPV1 and P2X7 receptor proteins in DRG of rats in each group by western blot;the double-labeled immunofluorescence experiment method was used to detect the co-expression of neuron TRPV1 and Neu N and satellite glial cell P2X7 receptor and GFAP in DRG of rats in each group;detect the expression of IL-1β,IL-10 and other inflammatory factors and p-p38,p-ERK1/2signaling pathway molecules in DRG by western blot;and use molecular docking to study whether A438079 has hydrogen bonding with P2X7 receptor and TRPV1.Results: 1.There was no significant difference in fasting blood glucose level and body weight of rats in the normal diet group(p>0.05).After feeding high-sugar and high-fat for four weeks and intraperitoneal injection of STZ,the fasting blood glucose level of the DNP model group was significantly higher than that of the control group(p<0.01)and the body weight was significantly lower than that of the control group(p<0.01).2.Behavioral results showed that the mechanical hyperalgesia and thermal hyperalgesia of DNP rats were lower compared with those of control group rats(p<0.01);there was no statistical difference between DNP+NS group and DNP group(p>0.05);at the end of the 8th week,the mechanical hyperalgesia and thermal hyperalgesia of rats in the DNP group injected with the P2X7 receptor antagonist A438079 intrathecally(DNP+A438079)were significantly higher than those in the DNP group(p<0.01).3.The qPCR results exhibited that noticeably lower levels of the TRPV1 and P2X7 m RNA were observed in the control group than in the DNP group(p<0.01),while lower levels of the TRPV1 and P2X7 m RNA were observed in the DRG in the DNP+A438079 group than in the DNP group(p<0.01;p<0.05).In contrast,no fundamental difference in TRPV1 and P2X7 proteins expression were observed between the control group and the DNP+A438079 group(p>0.05).4.Western blot results showed that noticeably lower levels of the TRPV1 and P2X7 protein were observed in the control group than in the DNP group(p<0.01),while lower levels of the TRPV1 and P2X7 protein were observed in the DRG in the DNP+A438079 group than in the DNP group(p<0.01).In contrast,no fundamental difference in TRPV1 and P2X7 protein expression were observed between the DNP+A438079 group and the control group(p>0.05).5.The immunofluorescence results exhibited that noticeably higher levels of TRPV1 with Neu N coexpression and GFAP with P2X7 receptor were observed in the DNP group than in the control group(p<0.01),while lower levels of coexpression were observed in the DNP+A438079 group than in the DNP group(p<0.01),but significant changes were not detected between the control group and the DNP+A438079 group.6.Western blot results exhibited that,the relative level of the IL-1β protein in the DRG from the DNP group was higher than that in the control group(p<0.01);while a lower level of IL-1β was observed in the DRG from the DNP+A438079 group than in the DNP group(p<0.05);but no significant difference was observed between the DNP+NS group and DNP group(p>0.05).In contrast,the relative level of the IL-10 protein in the DRG from the DNP group was lower than that in the control group(p<0.05);while a higher level of IL-10 was detected in the DRG in the DNP+A438079 group than in the DNP group(p<0.01);but no significant difference was observed between the DNP+NS group and DNP group(p>0.05).7.Western blot results exhibited that noticeably lower levels of the the p-p38 and p ERK1/2 protein were observed in the control group than in the DNP group(p<0.01),while lower levels of the the p-p38 and p ERK1/2 protein were observed in the DRG in the DNP+A438079 group than in the DNP group(p<0.01;p<0.05).In contrast,no fundamental difference in the p-p38 and p ERK1/2 protein expression were observed between the DNP+NS group and DNP group(p>0.05).8.The results of molecular docking show that the P2X7 receptor antagonist A438079 has hydrogen bonding to the P2X7 receptor,but not to TRPV1.Conclusion: The P2X7 receptor is involved in TRPV1-mediated DNP,and the p38 and ERK1/2 signaling pathways may play a role in its pathophysiological process. |
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